DESCRIPTION (provided by applicant Kaposi's Sarcoma-Associated Herpesvirus (KSHV) is the causative agent of Kaposi's Sarcoma (KS), one of the most common cancers in those infected with HIV. Like all herpesviruses, KSHV has a lytic phase, in which the virus actively replicates, and a latent phase, during which the virus remains quiescent and expresses only a limited set of genes. Among these genes are the viral microRNAs (miRNAs). miRNAs are short, 19-22 nucleotide long RNAs which post-transcriptionally suppress gene expression. Since KS tumors consist mainly of cells of endothelial origin which are latently infected with KSHV, and the viral miRNAs are expressed during latency, the role of the miRNAs in oncogenesis needs to be clarified. In order to understand the function of a given miRNA it is necessary to elucidate the genes which are targeted by it. We will determine which genes expressed in endothelial cells are targeted by the KSHV miRNAs using the High-Throughput Sequencing and Cross-Linking Immunoprecipitation (HITS-CLIP) method. In this procedure, live cells are exposed to UV light at 254 nm, which crosslinks protein with associated nucleic acid. The argonaute protein, which binds both miRNA and corresponding mRNA, is then immunoprecipitated with a specific antibody. Once the protein is digested the RNA can be purified, reverse-transcribed into cDNA, and sequenced. The sequences are aligned to the human genome in order to ascertain which genes are miRNA targets. Our laboratory has previously generated 12 KSHV mutant viruses, each of which lacks one of the 12 miRNAs encoded by KSHV. HITS-CLIP will be performed using Telomerase-Immortalized Vein Endothelial (TIVE) cells separately infected with either wild type KSHV or with two KSHV mutants lacking miR-K12-3 and miR-K12-11, respectively. These mutant viruses have previously been observed by our laboratory to be deficient in maintaining latency. In tandem, microarrays for human mRNAs will be performed on infected cells to see if the genes identified by HITS-CLIP to be targeted by KSHV miRNAs show a corresponding decrease in mRNA transcript level. In another set of experiments, TIVE cells will be separately infected with wild type and each of the 12 miRNA knockout viruses. Wild type KSHV is known to cause certain cellular phenotypic changes, and so the knockout viruses will be examined for the cellular phenotypes which they produce. This should indicate which host pathways and processes are affected by each of the KSHV miRNAs. Putative targeted genes identified by HITS-CLIP and microarray which are involved in these pathways can then be experimentally validated, thereby creating a complete picture of the biological role of each of the KSHV miRNAs and the mechanism behind it.